A Novel Polypyrimidine Tract-binding Protein Paralog Expressed in Smooth Muscle Cells
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.m210131200
ISSN1083-351X
AutoresClare Gooding, Paul R. Kemp, Christopher W. J. Smith,
Tópico(s)RNA modifications and cancer
ResumoPolypyrimidine tract-binding protein (PTB) is an abundant widespread RNA-binding protein with roles in regulation of pre-mRNA alternative splicing and 3′-end processing, internal ribosomal entry site-driven translation, and mRNA localization. Tissue-restricted paralogs of PTB have previously been reported in neuronal and hematopoietic cells. These proteins are thought to replace many general functions of PTB, but to have some distinct activities, e.g. in the tissue-specific regulation of some alternative splicing events. We report the identification and characterization of a fourth rodent PTB paralog (smPTB) that is expressed at high levels in a number of smooth muscle tissues. Recombinant smPTB localized to the nucleus, bound to RNA, and was able to regulate alternative splicing. We suggest that replacement of PTB by smPTB might be important in controlling some pre-mRNA alternative splicing events. Polypyrimidine tract-binding protein (PTB) is an abundant widespread RNA-binding protein with roles in regulation of pre-mRNA alternative splicing and 3′-end processing, internal ribosomal entry site-driven translation, and mRNA localization. Tissue-restricted paralogs of PTB have previously been reported in neuronal and hematopoietic cells. These proteins are thought to replace many general functions of PTB, but to have some distinct activities, e.g. in the tissue-specific regulation of some alternative splicing events. We report the identification and characterization of a fourth rodent PTB paralog (smPTB) that is expressed at high levels in a number of smooth muscle tissues. Recombinant smPTB localized to the nucleus, bound to RNA, and was able to regulate alternative splicing. We suggest that replacement of PTB by smPTB might be important in controlling some pre-mRNA alternative splicing events. heterogeneous nuclear ribonucleoproteins polypyrimidine tract-binding protein neurally enriched PTB brain enriched PTB smooth muscle PTB smooth muscle non-muscle rat aorta smooth muscle α-tropomyosin RNA recognition motif reverse transcription rapid amplification of cDNA ends green fluorescent protein group of overlapping clones The importance of post-transcriptional mechanisms of gene regulation has been emphasized by the relatively modest number of genes in the human genome (1Venter J.C.A. Myers M.D. Li Mural P.W. Sutton R.J. Smith G.G. Yandell H.O. Evans M. Holt C.A. Gocayne R.A. Amanatides J.D. Ballew P. Huson R.M. Wortman D.H. Zhang J.R. Kodira Q. Zheng C.D. Chen X.H. Skupski L. Subramanian M. Tho G. Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10387) Google Scholar, 2International Human Genome Sequencing Consortium Nature. 2001; 409: 860-921Crossref PubMed Scopus (17336) Google Scholar). Alternative splicing, RNA editing, and alternative translational initiation all allow for more than one protein isoform to be produced by individual genes. Alternative splicing is the most prevalent of the post-transcriptional mechanisms for producing protein isoforms. Conservative estimates predict that one- to two-thirds of human genes are alternatively spliced, and some of these genes have the potential to produce thousands of isoforms (reviewed in Refs. 3Black D.L. Cell. 2000; 103: 367-370Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 4Graveley B.R. Trends Genet. 2001; 17: 100-107Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 5Modrek B. Lee C. Nat. Genet. 2002; 30: 13-19Crossref PubMed Scopus (1039) Google Scholar, 6Roberts G.C. Smith C.W.J. Curr. Opin. Chem. Biol. 2002; 6: 375-383Crossref PubMed Scopus (110) Google Scholar, 7Maniatis T. Tasic B. Nature. 2002; 418: 236-243Crossref PubMed Scopus (593) Google Scholar). Regulation of alternative splicing involves the interaction of cellulartrans-acting factors with specific cis-acting regulatory elements within a target pre-mRNA (8Smith C.W.J. Valcárcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar, 9Caceres J.F. Kornblihtt A.R. Trends Genet. 2002; 18: 186-193Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). These regulatory interactions influence the recognition of splice sites by the splicing machinery. Such regulation can be positive, involving activator factors and enhancer sequences. Conversely, repressor proteins can mediate their influence via silencer elements. Although some model systems of regulated splicing involve the presence or absence of a single regulatory protein, the majority of examples appear to be more complex, with regulatory decisions being achieved by particular combinations of regulatory factors, each of which is expressed more widely than the splicing event that is being regulated (7Maniatis T. Tasic B. Nature. 2002; 418: 236-243Crossref PubMed Scopus (593) Google Scholar, 8Smith C.W.J. Valcárcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (747) Google Scholar, 9Caceres J.F. Kornblihtt A.R. Trends Genet. 2002; 18: 186-193Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). The heterogeneous nuclear ribonucleoproteins (hnRNPs)1 are a group of abundant and widespread nuclear proteins with diverse roles in pre-mRNA and mRNA function, including the regulation of alternative splicing (10Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Crossref PubMed Scopus (705) Google Scholar, 11Dreyfuss G. Kim V.K. Kataoka N. Nat. Rev. Mol. Cell Biol. 2002; 3: 195-205Crossref PubMed Scopus (1096) Google Scholar). Polypyrimidine tract-binding protein (PTB)1 (reviewed in Refs. 12Valcarcel J. Gebauer F. Curr. Biol. 1997; 7: R705-R708Abstract Full Text Full Text PDF PubMed Google Scholar and 13Wagner E.J. Garcia-Blanco M.A. Mol. Cell. Biol. 2001; 21: 3281-3288Crossref PubMed Scopus (302) Google Scholar), also known as hnRNP-I, is a prominent member of this family. PTB was originally identified as a potential splicing factor due to its ability to bind to polypyrimidine tracts at 3′-splice sites (14Garcia-Blanco M.A. Jamison S.F. Sharp P.A. Genes Dev. 1989; 3: 1874-1886Crossref PubMed Scopus (244) Google Scholar, 15Patton J.G. Mayer S.A. Tempst P. Nadal Ginard B. Genes Dev. 1991; 5: 1237-1251Crossref PubMed Scopus (289) Google Scholar, 16Gil A. Sharp P.A. Jamison S.F. Garcia-Blanco M.A. Genes Dev. 1991; 5: 1224-1236Crossref PubMed Scopus (224) Google Scholar). However, it was subsequently recognized to act as a splicing repressor at particular splice sites (17Ashiya M. Grabowski P.J. RNA (N. Y.). 1997; 3: 1-20PubMed Google Scholar, 18Carstens R.P. Wagner E.P. Garcia-Blanco M.A. Mol. Cell. Biol. 2000; 20: 7388-7400Crossref PubMed Scopus (123) Google Scholar, 19Chan R.C. Black D.L. Mol. Cell. Biol. 1997; 17: 4667-4676Crossref PubMed Google Scholar, 20Chou M.-Y. Underwood J.G. Nikolic J. Luu M.H.T. Black D.L. Mol. Cell. 2000; 5: 949-957Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 21Gooding C.G. Roberts G.C. Smith C.W.J. RNA (N. Y.). 1998; 4: 85-100PubMed Google Scholar, 22Jin W. McCutcheon I.E. Fuller G.N. Huang E.S. Cote G.J. Cancer Res. 2000; 60: 1221-1224PubMed Google Scholar, 23Lin C.-H. Patton J.G. RNA (N. Y.). 1995; 1: 234-245PubMed Google Scholar, 24Mulligan G.J. Guo W. Wormsley S. Helfman D.M. J. Biol. Chem. 1992; 267: 25480-25487Abstract Full Text PDF PubMed Google Scholar, 25Norton P.A. Nucleic Acids Res. 1994; 22: 3854-3860Crossref PubMed Scopus (71) Google Scholar, 26Perez I. Lin C.-H. McAfee J.G. Patton J.G. RNA (N. Y.). 1997; 3: 764-778PubMed Google Scholar, 27Singh R. Valcarcel J. Green M.R. Science. 1995; 268: 1173-1176Crossref PubMed Scopus (460) Google Scholar, 28Southby J. Gooding C. Smith C.W.J. Mol. Cell. Biol. 1999; 19: 2699-2711Crossref PubMed Google Scholar, 29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar, 30Zhang L. Liu W. Grabowski P.J. RNA (N. Y.). 1999; 5: 117-130Crossref PubMed Scopus (98) Google Scholar, 31Wagner E.J. Garcia-Blanco M.A. Mol. Cell. 2002; 10: 943-949Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). PTB also plays roles in nuclear pre-mRNA 3′-end processing (32Lou H. Helfman D.M. Gagel R.F. Berget S.M. Mol. Cell. Biol. 1999; 19: 78-85Crossref PubMed Scopus (128) Google Scholar, 33Moreira A. Takagaki Y. Brackenridge S. Wollerton M. Manley J.L. Proudfoot N.J. Genes Dev. 1998; 12: 2522-2534Crossref PubMed Scopus (133) Google Scholar), cytoplasmic internal ribosomal entry site-driven translation (34Kaminski A. Hunt S.L. Patton J.G. Jackson R.J. RNA (N. Y.). 1995; 1: 924-938PubMed Google Scholar), mRNA localization (35Cote C.A. Gautreau D. Denegre J.M. Kress T.L. Terry N.A. Mowry K.L. Mol. Cell. 1999; 4: 431-437Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), and regulation of mRNA stability (36Hamilton B.J. Genin A. Cron R.Q. Rigby W.F.C. Mol. Cell. Biol. 2003; 23: 510-525Crossref PubMed Scopus (69) Google Scholar). Consistent with these varied roles, PTB can shuttle between the nucleus and cytoplasm, but is predominantly localized in the nucleus (37Perez I. McAfee J.G. Patton J.G. Biochemistry. 1997; 36: 11881-11890Crossref PubMed Scopus (145) Google Scholar). The optimal RNA binding sequence for PTB (UCUU in a pyrimidine-rich context) is found within silencer elements that act by binding PTB (26Perez I. Lin C.-H. McAfee J.G. Patton J.G. RNA (N. Y.). 1997; 3: 764-778PubMed Google Scholar). These elements are often found within the 3′-splice site polypyrimidine tract; and in some cases, PTB acts by directly competing for binding to the polypyrimidine tract with the splicing factor U2AF65 (23Lin C.-H. Patton J.G. RNA (N. Y.). 1995; 1: 234-245PubMed Google Scholar, 27Singh R. Valcarcel J. Green M.R. Science. 1995; 268: 1173-1176Crossref PubMed Scopus (460) Google Scholar). However, PTB-binding sites are also found in other locations in the region of PTB-regulated exons, so it may also be able to inhibit splicing in other ways (reviewed in Ref. 13Wagner E.J. Garcia-Blanco M.A. Mol. Cell. Biol. 2001; 21: 3281-3288Crossref PubMed Scopus (302) Google Scholar). Consistent with its expression pattern, most PTB-mediated repression of specific exons is widespread. Regulated selection of the exons occurs in a small subset of tissues where the repressive action of PTB is either absent or in some way modulated. PTB exists in two major alternatively spliced isoforms, termed PTB1 and PTB4, which arise from skipping or inclusion, respectively, of exon 9, which encodes a 26-amino acid insert. A minor isoform, PTB2, is produced by inclusion of exon 9 using an internal 3′-splice site, giving a 19-amino acid insert. In at least one case, these isoforms have differential activity, with PTB4 being more repressive upon α-tropomyosin exon 3 than PTB2 or PTB1 (29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar). In addition to the PTB isoforms, at least two paralog genes, nPTB/brPTB and ROD1, with ∼70% amino acid identity to PTB have been identified. nPTB/brPTB (38Polydorides A.D. Okano H.J. Yang Y.Y.L. Stefani G. Darnell R.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6350-6355Crossref PubMed Scopus (192) Google Scholar, 39Markovtsov V. Nikolic J.M. Goldman J.A. Turck C.W. Chou M.-Y. Black D.L. Mol. Cell. Biol. 2000; 20: 7463-7479Crossref PubMed Scopus (248) Google Scholar) is expressed predominantly in neuronal cells, whereas ROD1 is expressed mainly in hematopoietic cells (40Yamamoto H. Tsukahara K. Kanaoka Y. Jinno S. Okayama H. Mol. Cell. Biol. 1999; 19: 3829-3841Crossref PubMed Scopus (90) Google Scholar). Cells expressing nPTB or ROD1 tend to express less PTB; and in the case of the alternative N1 exon of c-src, nPTB is less repressive than PTB, contributing to the neuronal selection of the N1 exon (39Markovtsov V. Nikolic J.M. Goldman J.A. Turck C.W. Chou M.-Y. Black D.L. Mol. Cell. Biol. 2000; 20: 7463-7479Crossref PubMed Scopus (248) Google Scholar). The effect of nPTB on other splicing events is similar to that of PTB (29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar). Although no functional data have been reported on ROD1, a reasonable proposal is that, in neuronal or hematopoietic cells expressing nPTB or ROD1, many PTB-repressed splicing events will be unaffected, whereas a specific subset will be altered. Alterations in the expression of the alternatively spliced isoforms of PTB or in the expression of paralog genes therefore provide one way in which PTB activity can be modulated. We have been investigating two alternative splicing events that are regulated in smooth muscle (SM) cells. In α-actinin, a SM-specific exon is repressed by PTB in non-SM cells, leading to inclusion of the mutually exclusive alternative non-muscle (NM) exon (see Fig.1A) (28Southby J. Gooding C. Smith C.W.J. Mol. Cell. Biol. 1999; 19: 2699-2711Crossref PubMed Google Scholar, 41Waites G.T. Graham I.R. Jackson P. Millake D.B. Patel B. Blanchard A.D. Weller P.A. Eperon I.C. Critchley D.R. J. Biol. Chem. 1992; 267: 6263-6271Abstract Full Text PDF PubMed Google Scholar). In α-tropomyosin (α-TM), exon 2 is included only as a result of repression of the mutually exclusive exon 3 in SM cells (see Fig. 1C) (42Gooding C. Roberts G.C. Moreau G. Nadal Ginard B. Smith C.W.J. EMBO J. 1994; 13: 3861-3872Crossref PubMed Scopus (92) Google Scholar). This repression is mediated in part by high affinity PTB-binding sites on either side of exon 3 (21Gooding C.G. Roberts G.C. Smith C.W.J. RNA (N. Y.). 1998; 4: 85-100PubMed Google Scholar, 26Perez I. Lin C.-H. McAfee J.G. Patton J.G. RNA (N. Y.). 1997; 3: 764-778PubMed Google Scholar). In vitro splicing experiments have shown that PTB mediates a low level of exon 3 repression in non-muscle extracts (29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar). However, in vivo, full repression is observed only in SM cells. In this respect, PTB-mediated repression of α-TM exon 3 differs from all other characterized splicing events regulated by PTB. In an attempt to understand how the tropomyosin and actinin splicing events are regulated, we investigated the expression of PTB isoforms in dedifferentiating rat aorta smooth muscle (RASM) cells. We found no change in the ratio of PTB1 and PTB4. However, in a number of SM tissues, we detected expression of a novel PTB paralog that is distinct from PTB, nPTB/brPTB, and ROD1. We refer to the new paralog as smPTB due to its initial identification and high levels of expression in SM tissues. smPTB is ∼70% identical to PTB and has additional 36- and 22-amino acid inserts in two of the linker regions separating RNA recognition motif (RRM) domains. Recombinant smPTB binds RNA in vitro and has splicing inhibitory activity. We propose that expression of smPTB may play a role in switching subsets of alternative splicing events in SM and other cells. Primary RASM cells were isolated by enzymatic dispersion and cultured as described (43Kemp P.R. Grainger D.J. Shanahan C.M. Weissberg P.L. Metcalfe J.C. Biochem. J. 1991; 277: 285-288Crossref PubMed Scopus (24) Google Scholar,44Shanahan C.M. Weissberg P.L. Metcalfe J.C. Circ. Res. 1993; 73: 193-204Crossref PubMed Scopus (318) Google Scholar). Total RNA from SM cells and tissues was made using TRI reagent (29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar). Splicing patterns of the different genes were analyzed by reverse transcription (RT)-PCR. Oligo(dT) was used for reverse transcription by avian myeloblastosis virus reverse transcriptase (45Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). PCR was carried out with a 32P-end-labeled primer using a hot start of 92 °C for 3 min, followed by enzyme addition at 80 °C. Thirty cycles were carried out at 94 °C for 30 s, annealing temperature (variable) for 30 s, and 72 °C for 60 s, with a final extension at 72 °C for 2 min. A 55 °C annealing temperature was used for primer sets 5′-vin/3′-vin, TM1/TM4, P30/31 (smPTB + PTB) (see Fig. 4), P37/Pd3′2 (smPTB + PTB) (see Fig.2), P37/P38 (PTB-specific), and P12/P38 (human PTB plasmid markers). A 58 °C annealing temperature was used for primer set P32/P33 (smPTB-specific). A 60 °C annealing temperature was used for primer set EF1a5′/3′-act. In each case, the 5′-primer is stated first. For the PTB plasmid marker PCR, 10% Me2SO was included. Products were analyzed on 4% denaturing polyacrylamide gels. The sequences of the oligonucleotides used for PCR are as follows: EF1a5′, 5′-ATCAGCCAGGAACAGATG-3′; 3′-act, 5′-ACATGAAGTCGATGAAGGCCTG-3′; 5′-vin, 5′-GGTGATTAACCAGCCAATGATGAT-3′; 3′-vin, 5′-CTTCACAGACTGCATGAGGTT-3′; TM1, 5′-CGAGCAGAGCAGGCGGAG-3′; TM4, 5′-CAGAGATGCTACGTCAGCTTCAGC-3′; P12, 5′-AAGAGCCGTGACTACACACGC-3′; P30, 5′-GACCTGCCCTC(A/T)G(A/G)(T/A)GACAG-3′; P31, 5′-GCGTTCTCCTTC(C/T)TGTTGAACA-3′; P33, 5′-TCGGTGCACATCGCCATAGGCA-3′; P37, 5′-AAGAGCCGAGACTACACACGC-3′; P38, 5′-GAGGCTTTGGGGTGTGACTCT-3′; and Pd3′2, 5′-TTGCCGTCCGCCATCTGCACTA-3′.Figure 2Novel PTB in differentiated SM cells.A, alternative splicing of PTB exons 9 and 11. Inclusion of exon 9 produces PTB4, whereas skipping leads to PTB1. Skipping of exon 11 produces mRNAs that encode truncated isoforms PTB1tr and PTB4tr. The positions of primers P37, P38, and Pd3′2 used for PCR inB and C are indicated. B, RT-PCR of RNA harvested from RASM cells at day 0 (0) or day 4 (4) or from PAC-1 cells. PCR primers P37 and P38 correspond to PTB exons 8 and 11, respectively. C, same asB, but using primers P37 and Pd3′2, corresponding to PTB exons 8 and 12, respectively, which also detect the novel smPTB paralog. D, same as B and C, but using primers specific for the smPTB paralog. Tissues from the uterus and vas deferens were also tested with smPTB-specific primers. Size markers are either HaeIII ØX174 (M lanes; sizes are indicated in bp) or PCR products from a mixture of PTB1, PTB2, and PTB4 plasmids.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Constructs were prepared by standard cloning techniques (45Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Full-length smPTB and PTB were cloned into vectors forin vivo transfection (pCMVSPORT), localization (pEGFPN1 or pEGFPC1), and overexpression in Escherichia coli (pET21). Rapid amplification of cDNA ends (RACE) was used to amplify smPTB in two halves using gene-specific primers from day 0 rat aorta RNA. 3′-RACE (45Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) was carried out as a first round with oligo(dT) and P32 (see below) as the forward gene-specific primer (30 cycles of 94 °C of 30 s, 55 °C for 30 s, and 72 °C for 60 s). This was followed by nested PCR with a forward gene-specific primer (P10) under the same cycle conditions. For 5′-cDNA, SMART RACE (Clontech) was used. Reverse transcription was carried out using Moloney murine leukemia virus reverse transcriptase with oligo(dT) plus a SMART oligonucleotide (P77). The first round of PCR was carried out (30 cycles of 94 °C for 30 s, 58 °C for 30 s, and 68 °C for 60 s) with a 5′-primer mixture at a ratio of 5:1 P79/P78 plus the gene-specific 3′-reverse primer P33 (see above). Advantage GC2 polymerase (Clontech) was used. This was followed by 25 cycles (94 °C for 30 s, 60 °C for 30 s, and 68 °C for 2 min) of nested PCR with a 5′-primer (P80) and a gene-specific primer (P11 or P41). The oligonucleotides used for RACE were as follows: P10, 5′-TTCCTCAAGCTGCAGGCTTGGCCA-3′; P11, 5′-GGGAGCCTGAGGTGACCATTGAGC-3′; P32, 5′-ATGACAGTCAGCCCTCTCCGGTC-3′; P41, 5′-GAGCTGAGGCCATGTTCTGGACCTGGACCGGA-3′; P77, 5′-AAGCAGTGGTAACAACGCAGAGTACGCGGG-3′; and P80, 5′-AAGCAGTGGTAACAACGCAGAGT. The sequence of the complete rat smPTB open reading frame has been deposited in the GenBankTM/EBI Data Bank (accession number AY223520). 35S-Labeled sense and antisense RNA probes corresponding to mouse smPTB amino acids 100–182 were transcribed in vitro and hybridized to day 10, 14, and 15 mouse embryo sections as described (46Ellis P.D. Chen Q. Barker P.J. Metcalfe J.C. Kemp P.R. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1912-1919Crossref PubMed Scopus (55) Google Scholar). PAC-1 or HeLa cells grown on coverslips were transiently transfected, using LipofectAMINE (Invitrogen), with PTB or smPTB tagged with green fluorescent protein (GFP) at the C or N terminus. After 24 or 48 h, the coverslips were inverted on a microscope slide, and GFP was visualized using a Zeiss fluorescent microscope with an MC100 camera attached for photographs. C-terminally His6-tagged recombinant proteins were expressed and purified as previously described (46Ellis P.D. Chen Q. Barker P.J. Metcalfe J.C. Kemp P.R. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1912-1919Crossref PubMed Scopus (55) Google Scholar) with one modification for smPTB. To solubilize smPTB, it was necessary to re-extract the pellet after lysis using the non-detergent sulfobetaine (1 m; NDSB-201) in lysis buffer (47Goldberg M.E. Expert-Bezancon N. Vuillard L. Rabilloud T. Folding Des. 1995; 1: 21-27Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). High specific activity [α-32P]UTP-labeled RNA probes (20 fmol) were incubated with recombinant protein in 12 mm Hepes, pH 7.9, 100 mm KCl, 3% glycerol, 0.1 mm EDTA, 0.3 mm dithiothreitol, and 50 μg/ml E. colirRNA for 20 min at 30 °C. For competitive binding experiments, PTB4 and smPTB were premixed before addition of the RNA. Heparin was added to 0.25 mg/ml, and the reaction was left for 5 min at room temperature. Samples were irradiated on ice at 254 nm in a Spectrolinker cross-linker with a controlled energy dose of 1.92 mJ. RNA was digested with RNases T1 (1 unit/μl) and A (0.4 mg/ml), and the samples were run on SDS-polyacrylamide gels. PAC-1 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Transient transfection was carried out using LipofectAMINE; total RNA was isolated using TRI reagent; and RT-PCR for α-actinin was carried out as previously described (29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar). In view of the evidence for the involvement of PTB in controlling alternative splicing of α-TM and α-actinin and of the differential activity of the alternatively spliced PTB isoforms upon α-TM splicing (29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar), we decided to analyze expression of PTB in a well characterized model cell system in which these events are regulated. Freshly isolated RASM cells are initially highly differentiated, but, over the course of 4–6 days in culture, become dedifferentiated and cease to express a number of genes associated with the differentiated contractile state (43Kemp P.R. Grainger D.J. Shanahan C.M. Weissberg P.L. Metcalfe J.C. Biochem. J. 1991; 277: 285-288Crossref PubMed Scopus (24) Google Scholar). We analyzed alternative splicing of α-TM, α-actinin, and vinculin/meta-vinculin in RNA from RASM cells at day 0 or 4 in culture and from the PAC-1 pulmonary artery SM cell line (Fig.1). RT-PCR analysis of α-actinin splicing indicated that, at day 0, the major product included the SM exon, with a small amount containing the larger NM exon (Fig.1A). A small quantity of product corresponding to skipping of both the NM and SM exons was also observed. Like the SM isoform, this "double-skipped" product, which has not been reported before, would result in a nonfunctional EF-hand domain. By day 4, there were roughly equal amounts of the SM and NM isoforms, with a decrease in the amount of the skipped product. The cultured PAC-1 cells were similar to the day 4 cells, but with more NM than SM inclusion and no double-skipped product. RT-PCR analysis of vinculin (Fig.1B) showed that the meta-vinculin isoform was expressed as a minor isoform only in day 0 RASM cells and was undetectable in day 4 and PAC-1 cells. Mutually exclusive splicing of α-TM exons 2 and 3 produced products of identical size. To differentiate them, the radiolabeled PCR products were digested withXhoI, which cuts within exon 2 to produce a 145-nucleotide product, or with PvuII, which cuts exon 3 products to produce a 150-nucleotide band (Fig. 1C). Double digests showed that the PCR product could be fully digested by both enzymes. The almost complete XhoI digestion and PvuII resistance of the day 0 PCR product showed that fully differentiated RASM cells predominantly expressed the exon 2-containing α-TM isoform. By day 4, PvuII digested a greater proportion of the PCR product compared with XhoI, indicating a substantial switch toward inclusion of exon 3 instead of exon 2. In comparison, in cultured PAC-1 cells, the majority of α-TM RNA contained exon 3. PAC-1 cells commonly show a greater degree of regulated splicing than observed here (21Gooding C.G. Roberts G.C. Smith C.W.J. RNA (N. Y.). 1998; 4: 85-100PubMed Google Scholar, 42Gooding C. Roberts G.C. Moreau G. Nadal Ginard B. Smith C.W.J. EMBO J. 1994; 13: 3861-3872Crossref PubMed Scopus (92) Google Scholar, 48Roberts G.C. Gooding C. Mak H.Y. Proudfoot N.J. Smith C.W.J. Nucleic Acids Res. 1998; 26: 5568-5572Crossref PubMed Scopus (151) Google Scholar), but they served as a useful undifferentiated control sample. Taken together, the data indicate that the three alternative splicing events analyzed in RASM cells showed a substantial switch toward the non-SM pattern after 4 days in culture. Having observed the switch in alternative splicing of α-TM and α-actinin, both of which are regulated by PTB, we next analyzed expression of the PTB isoforms. RT-PCR was carried out using primers P37 and P38, which correspond to exons 8 and 11, respectively. This analysis allows the detection of alternative splicing of exon 9, which gives rise to the PTB1 and PTB4 isoforms. Unlike α-TM, α-actinin, and vinculin, alternative splicing of the PTB1 and PTB4 isoforms showed no significant changes between the day 0 and 4 RASM and PAC-1 samples (Fig.2B). Therefore, despite the fact that PTB4 has been shown to be a more active repressor of α-TM exon 3 compared with PTB1 (29Wollerton M. Gooding C. Robinson F. Brown E. Jackson R. Smith C.W.J. RNA (N. Y.). 2001; 7: 819-832Crossref PubMed Scopus (114) Google Scholar), changes in the ratio of the PTB isoforms do not cause the switch in α-TM and α-actinin splicing in dedifferentiating RASM cells. RT-PCR was not carried out under conditions that would allow quantitative analysis of absolute levels of expression. Nevertheless, we consistently observed that the levels of PTB products appeared to be lower in day 0 cells than in day 4 cells. Further analysis of PTB expression using primers P37 and Pd3′2, which prime within exons 8 and 12, respectively, produced a strikingly different result. A novel band (labeled smPTB in Fig.2, C and D) larger than PTB4 was the major PCR product in day 0 samples, but not in day 4 or PAC-1 samples. Cloning and sequencing of this PCR product showed that it was derived from a PTB-related gene that was distinct from PTB and the known paralogsROD1 (40Yamamoto H. Tsukahara K. Kanaoka Y. Jinno S. Okayama H. Mol. Cell. Biol. 1999; 19: 3829-3841Crossref PubMed Scopus (90) Google Scholar) and nPTB (38Polydorides A.D. Okano H.J. Yang Y.Y.L. Stefani G. Darnell R.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6350-6355Crossref PubMed Scopus (192) Google Scholar, 39Markovtsov V. Nikolic J.M. Goldman J.A. Turck C.W. Chou M.-Y. Black D.L. Mol. Cell. Biol. 2000; 20: 7463-7479Crossref PubMed Scopus (248) Google Scholar). We refer to this new PTB paralog as smPTB due to its high expression in a number of SM tissues (see below). RT-PCR using smPTB-specific primers P32 and P33 confirmed that it was expressed in day 0 RASM cells, but not in day 4 or cultured PAC-1 cells (Fig. 2D). smPTB was also expressed in other SM tissues such as the uterus and vas deferens. At the time that we identified smPTB, no corresponding sequences could be identified by BLAST searches of available expressed sequence tag or genomic data bases. However, using 3′- and 5′-RACE, full-length smPTB cDNA was isolated from day 0 RASM cell RNA. The open reading frame encodes a 588-amino acid protein with a predicted molecular mass of 63.7 kDa and with 53–74% amino acid identity to PTB, nPTB, and ROD1 (Fig.3). Pairwise BLAST analyses showed that smPTB is more closely related to PTB than to either of the other genes. Subsequent to cloning the full-length smPTB cDNA, the corresponding rat gene sequence was identified using the ENSEMBL Trace Database, whereas the mouse gene was identified by BLAST analysis of annotated mouse genomic data bases and was located in contig 132920, corresponding to chromosome X A1.1. Both the rat and mouse genes are intronless. The mouse gene contains three possible polyadenylation addition signals giving a message size of 4.16, 5.09, or 6.53 kb. As determined by Northern blot analysis, the size of the rat smPTB mRNA is close to 6 kb (data not shown). smPTB has the same overall structural organization as PTB (Fig. 3), with four RRM domains and the same unusu
Referência(s)